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Socio-environmental and endocrine influences on developmental and
caste-regulatory gene expression in the eusocial termite Reticulitermes flavipes
BMC Molecular Biology 2010, 11:28 doi:10.1186/1471-2199-11-28
Matthew R Tarver (Matt.Tarver@ARS.USDA.GOV)
Xuguo Zhou (email@example.com)
Michael E Scharf (firstname.lastname@example.org)
28 October 2009
23 April 2010
23 April 2010
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Socio-environmental and endocrine influences on
developmental and caste-regulatory gene expression
in the eusocial termite Reticulitermes flavipes
Matthew R. Tarver1'2, Xuguo Zhou'3 and Michael E. Scharf'
Department of Entomology and Nematology, University of Florida, Gainesville FL,
2 Present Address: Formosan Subterranean Termite Research Unit, USDA-ARS-SRRC,
New Orleans LA, USA
3 Present Address: Department of Entomology, University of Kentucky, Lexington,
Strict regulation of caste differentiation, at the molecular level, is thought to be important to
maintain social structure in insect societies. Previously, a number of extrinsic and intrinsic
factors have been shown to influence caste composition in termite colonies. One important
factor is the influence of nestmates; in particular, soldier termites are known to inhibit
hormone-dependent worker-to-soldier differentiation. However, soldier influences on nestmates
at the molecular level are virtually unknown. Here, to test the hypothesis that soldiers can
influence nestmate gene expression, we investigated the impact of four treatments on whole-
body gene expression in totipotent Reticulitermesflavipes workers: (i) juvenile hormone III
(JHIII; a morphogenetic hormone), (ii) soldier head extracts (SHE), (iii) JHIII+SHE, and (iv)
Using quantitative-real-time PCR we determined the expression patterns of 49 previously
identified candidate genes in response to the four treatments at assay days 1, 5, and 10. Thirty-
eight total genes from three categories (chemical production / degradation, hemolymph protein,
and developmental) showed significant differential expression among treatments. Most
importantly, SHE and live soldier treatments had a significant impact on a number of genes
from families known to play roles in insect development, supporting previous findings and
hypotheses that soldiers regulate nestmate caste differentiation via terpene primer pheromones
contained in their heads.
This research provides new insights into the impacts that socio-environmental factors (JH,
soldiers, primer pheromones) can have on termite gene expression and caste differentiation, and
reveals a number of socially-relevant genes for investigation in subsequent caste differentiation
Phenotypic plasticity can be described as the production of variable phenotypes
from a single genotype based on conditions encountered throughout an organism's
development . Phenotypic plasticity can be divided into gradual or discrete
polyphenisms. Reaction norms are phenotypically graded responses to environmental
factors. Polyphenisms, occur when two or more discrete alternative phenotypes occur
without intermediate forms .
Social insects have evolved to produce and use multiple alternate phenotypes
(i.e., polyphenism) to accomplish a wide range of tasks within their colonies. Castes are
phenotypically and behaviorally discrete individuals that cooperate to perform colony
tasks . Termites are hemimetabolous social insects that utilize castes to meet various
needs within the colony. Most termite colonies are made up of three distinct castes:
workers, soldiers, and reproductive . All termite eggs, except when a rare genetic
component might be involved , are considered totipotent, and most evidence
supports that castes differentiate based on gene expression responses to intrinsic and
extrinsic factors. The research presented here examined gene expression responses in
worker termites to both intrinsic and extrinsic factors.
All castes except soldiers and reproductive retain the ability to molt, while
soldiers and reproductive are considered terminally developed . Caste
differentiation can proceed along two routes; imaginal (winged) or apterous (wingless).
The first developmental branch is the point at which larvae differentiate into apterous
workers or imaginal nymphs. Nymphs can either (i) regress into worker-like
"pseudergates", (ii) differentiate into fully winged and eyed adult alates that disperse
and found new colonies, or (iii) differentiate into winged and eyed non-dispersive
brachypterous reproductive that serve as supplemental reproductive within the
colony. Workers are totipotent in that they can (i) undergo status quo worker-to-worker
molts, (ii) differentiate into soldiers (after passing through an intermediate presoldier
stage), or (iii) differentiate into apterous and eyeless neotenic reproductive that serve
supplementary reproductive roles [6-8].
The entire complement of intrinsic and extrinsic factors that dictate each of the
developmental switches in termites, and how they interact, are yet to be fully
understood. Examples of intrinsic factors include juvenile hormone (JH), storage
proteins, and nutrition; whereas examples of extrinsic factors include primer
pheromones, temperature, food quality, nestmates (soldiers and reproductives, and
Phenotypic divergence from the worker to the soldier caste can be mediated by
multiple JH-related factors. For example, elevated JH titers in workers are correlated
with presoldier differentiation [20-22]. Additionally, the presence of soldiers has been
shown to inhibit the formation of new soldiers, implying that soldier termites produce
inhibitory factors that cause reduced responsiveness to JH or reduced JH biosynthesis
in nestmates [15,16,23,24]. This inhibition is presumed to be caused by soldier-derived
primer pheromones [10,25,26]. Primer pheromones are defined as chemical messengers
that are passed among individuals and trigger physiological responses in recipients
. Recently, R. flavipes soldier head extracts (SHE), when applied in combination
with juvenile hormone III (JH III), were found to enhance presoldier production
compared to JH III alone . Two major components of R. flavipes SHE are y-
cadinene and its aldehyde y-cadinenal; they represent the first candidate primer
pheromones to be identified from termites . Interestingly, SHE alone does not
impact presoldier formation . Also, while the SHE blend is active at influencing
JH-dependent presoldier differentiation, the individual impacts of its constituents and
whether they are being actively released or absorbed has yet to be determined.
Functional genomics is a powerful approach for elucidating the functions of
genes, including genes that mediate pheromone and hormone action . Transcript
levels generally correlate with the physiological demand for the product they produce;
thus, changes in transcript abundance can reveal genes that are most important in
relation to a stimulus . Such an approach has been used to elucidate the chemical
ecology of the bark beetle (Ips pini) [28-31] and the honeybee (Apis mellifera) .
Similarly, the use of functional genomics in studies of termite caste regulation can help
to better understand potential primer pheromone function as well as the influences of
intrinsic and extrinsic factors on caste differentiation.
The central goal of this research was to use a functional genomics approach to
identify candidate caste-regulatory genes from R. flavipes workers that potentially
mediate hormonal and soldier primer pheromone signaling. Four treatments were
tested on isolated groups of worker termites: (i) JH III alone, (ii) soldier head extract
(SHE) alone, (iii) JH III + SHE, and (iv) live soldiers. These treatments represent key
intrinsic and extrinsic / socio-environmental factors that are thought to impact soldier
development in totipotent workers. Our central hypothesis was that these four
treatments will be associated with the differential expression of key genes through time,
and that key responsive genes will play significant roles in meditating caste
differentiation and/or caste-regulatory signaling. Our approach involved determining
the impacts of the four treatments on both phenotypic caste differentiation and the
expression of forty-nine candidate and reference genes during the first 10 days of
differentiation from worker to presoldier, with subsequent validation of reference genes
and post-hoc analyses to identify genes with significant differential expression among
treatments. Here, we identify and discuss a number of responsive genes from three
categories (chemical production / degradation, hemolymph protein, and developmental)
with significant links to caste differentiation.
Phenotypic bioassays (Fig. 1A) showed that the combination of JH III + SHE
significantly increased presoldier development when compared to JH III alone. A two-
way ANOVA and adjusted LS means were used for analysis (whole model F=24.092,
df=14, P<0.0001; treatment F= 54.32, df=4, P<0.0001; colony F=24.140, df=2,
P<0.0001; treatment*colony F=11.513, df=8, P<0.0001). Variation was observed
between the different colonies tested, with Colony 1 showing the greatest presoldier
induction response to JH III (40%) and JH III+SHE (80%). But, as seen in previous
research , the overall trend was the same in that JH III+SHE increased presoldier
differentiation compared to JH III alone and no presoldiers formed in the acetone-
treated controls, SHE-alone treatments, or live soldier treatments (Fig. 1A).
Because no phenotypic effects were observed with live soldier treatments in the
small-format dish assays noted above, we conducted post-hoc presoldier induction
assays using larger groups of workers (Fig. 1B). Our objective was to determine if
soldiers could inhibit natural presoldier formation in the absence of ectopic JH, using
greater numbers of workers (100 termites) over a longer period of time (30 days). Two
treatments were tested: (1) 100 workers + 0 soldiers, and (2) 90 workers + 10 soldiers.
Interestingly, no presoldiers formed after 30 d. in treatments that included 10% soldiers
at the beginning of assays; and conversely, presoldiers appeared only in the treatments
that included 100% workers at the beginning of assays. This finding verifies that R.
flavipes soldiers are capable of inhibiting worker-to-presoldier differentiation, and
provides evidence that is directly supportive of the soldier and SHE impacts on gene
expression presented below.
Reference gene selection
To accurately determine relative gene expression in totipotent workers, we chose
three reference genes that had stable expression across all treatments and colonies
(Stero-1, LIM, and Mev-1). These reference genes were selected by comparing the
standard deviation of the raw Ct values for all 49 genes across treatments (Additional
file 1: Table Si). This determination is important because it allows normalization of the
expression of target genes (n=46) to reference genes (n=3) that have stable expression
across all treatments and colonies.
Gene expression overview
All target and reference genes investigated in this study have been annotated
based on significant translated identity to insect sequences deposited in the Genbank nr
and EST databases. Full-length gene names are provided in Additional file 2: Table S2.
All reported gene expression data represent the average of three independently sampled
and replicated R. flavipes colonies. Gene expression changes in response to all
treatments were determined via qRT-PCR. To identify genes with significant
differential expression across treatments, two-way ANOVAs were used with adjusted
LS means and FDR correction on normalized CT (ACT) values (Additional files
3,4,5,6: Tables S3, S4, S5, S6). Additionally, gene expression at three days (1, 5 and
10) was analyzed separately using the ANOVA procedure noted above. For a large
proportion of the genes tested there was a significant colony effect. This was to be
expected because (i) there was also a significant colony effect in the phenotypic
bioassay and (ii) the colonies tested each have different mitochondrial haplotypes (see
later). Colony effects were compensated for by using adjusted LS means in the
To easily visualize gene expression responses, genes showing significant
expression changes across treatments were organized by day into heat maps (Fig.
2,3,4). Genes with similar expression profiles are horizontally clustered together. By
clustering genes in this manner we are able to identify groups of genes that respond
similarly and putatively belong to the same gene networks (also see Additional file 7:
Gene expression: day 1
As shown in the Day 1 heat map (Fig. 2), 17 out of the 46 genes that were tested
showed significant differences in their expression across treatments (Additional file 4:
Table S4). Day 1 receives focus here because we presume Day 1 responsive genes to
be important immediate-early responders. Three main clusters of genes were identified,
with sub groupings of genes in some clusters. Genes in group IIB overall were affected
by SHE and live solder treatments, with IIB2ii genes Carbx-1, 3!v, ,Win. B-actin, fl-tube,
R-Pro, ATPase, and HMG all being down-regulated with live soldiers. Genes in group
IIB1, NADH and nanos, were up-regulated in live soldier treatments. Group IIB2i
genes Hex-2 and 18s were down-regulated in SHE treatments. The P450 protein coding
genes in group IIA, CYP15FI, CYP4C48, CYP6G?, and CYP4C47 were down
regulated with JH III and JH III+SHE treatments, while group I genes, CYP4C46 and
CYP4U3, were up-regulated with JH III. These Day 1 results reveal a number of early
response genes in totipotent workers that are both up and down-regulated in response to
the different treatments. Perhaps most importantly, a number of P450 genes that may
play roles in semiochemical or hormone processing were differentially expressed
among treatments at this early time point.
Gene expression: day 5
Five days into assays, 23 genes showed significant differential expression among
the five treatments (Fig. 3, Additional file 5: Table S5). A larger number of genes
showed significant variation in expression at this point compared with Days 1 and 10,
with the majority of the genes showing down-regulated responses to most treatments.
Genes in group IIB2iib3, CYP4C44vl, broad, and APO had a slight expression
increase with JH, while being down-regulated with SHE and live soldier treatments.
Group IIB2iib2 genes, CoxII, HSP, and Shp displayed an up-regulation with live
soldier treatments. Genes SH3, NADH and CYP15F1, in group IIB2iibl, were down-
regulated with JH+SHE and SHE treatments. Group IIB2iia genes, Famet-2, Carbx-1,
CYP4U3, Carbx-2, and To-F were all down-regulated with live soldier treatments. Bic
and nanos, in group IIB2i, were down-regulated with JH III, JH III+SHE and SHE
treatments. Genes that clustered into group IIB1, Hex-2, Hex-1, and CYP4C46 were
up-regulated with JH III and JH+SHE treatments. Finally, two hemolymph protein
coding genes, Vit-1 (IIA) and Vit-2 (I) were up-regulated with JH III and JH+SHE and
down-regulated with SHE and live soldier treatments. Five days into assays represents
the middle of the worker-to-solder differentiation process . Therefore, genes
identified at this time point could be playing mid-level signaling roles in the caste
differentiation cascade. The hemolymph protein coding genes Vit-1, Vit-2, Hex-1 and
Hex-2, have been linked to caste differentiation in past research in termites and honey
bees [17,18,33-38]. Thus, their differential expression during the worker-to-presoldier
differentiation process was expected, and serves to validate our approach for
determining gene expression during differentiation.
Gene expression: day 10
On the last day investigated (Day 10) nineteen genes showed significant variation
in expression across treatments (Fig. 4, Additional file 6: Table S6). Live soldier effects
were not investigated at this time point due to limitations imposed by the 96-well PCR
plate format and an inability to include all treatments for individual genes on a single
plate. The group II genes Epox-1 and Vit-2 were up-regulated with JH III and JH+SHE
treatments. Genes in group IB3iib, CYP15F1, Shp, and Tro-1 were down-regulated
with JH+SHE treatment, while Hex-1 and To-F (IB3iia) were down regulated with JH
III and JH+SHE treatments. The putative ribosomal RNA coding 18s gene was down-
regulated in live soldier treatments (IB3i). Group IB2ii genes CYP4U3, 28s, and
CYP4C46 were up-regulated with JH III but down-regulated with JH+SHE treatment,
while genes in group IB2i, Lprs, Famet-1, and NADH were down-regulated with JH III.
Genes that clustered in group IB1, Myosin, APO, and broad were up-regulated with
JH+SHE treatment. Finally group IA genes, Carbx-1 and SH3, were down-regulated
with JH+SHE treatment. These Day 10 results reveal a number of potential late
responding genes that are both up- and down-regulated in response to the different
treatments. Thus, these late responding genes likely are part of multiple pathways that
are involved in the later stages of the worker-to-presoldier differentiation process.
Uniformly responsive genes and hierarchical clustering
Across days 1, 5 and 10, four genes showed consistent, significant differential
expression: CYP15FI, CYP4C46, CYP4U3, and NADH. This finding suggests that
these four genes are of broad general importance in worker-to-soldier caste
differentiation and / or caste regulation / homeostasis.
Finally, gene expression results were hierarchically (vertically) clustered by
treatment across days based on the expression patterns of all genes (Fig. 5a,b,c).
Results for Day 1 and 5 are similar with control and live soldier treatments clustering
together, and JH III and JH+SHE treatments clustering together. Day 10 results show a
different clustering pattern in which control and SHE treatments cluster together, and
the JH and JH+SHE treatments show a more distant relationship. These results suggest
that effects of the different treatments on genes and gene networks are not temporally
fixed, but change through time.
Social organisms, including hemimetabolous lower termites like R. flavipes,
utilize phenotypic plasticity to achieve caste polyphenism and division of labor.
Because all termite colony members share essentially the same genetic background,
they rely on differential gene expression for caste differentiation . The development
of termites along alternate caste pathways is regulated by a number of interacting
intrinsic and extrinsic factors (e.g., ); however, detailed global gene expression
responses through the differentiation process have been lacking, and no prior studies
have investigated nestmate or primer-pheromone-responsive gene expression in
This study correlates clear phenotypic effects of R. flavipes hormones,
semiochemicals, and social treatments with patterns of gene expression and reveals
potentially important candidate caste-regulatory genes. Changes in expression of
several genes having homology to other well-characterized developmental and
hormone / semiochemical biotransformation genes were detected in association with
the different treatments. Several gene networks apparently important in caste
differentiation and social interactions were also identified.
The model bioassay system used here induces changes in phenotype, and gene /
protein expression, and has been used repeatedly to monitor and elucidate mechanisms
of caste differentiation, specifically the worker-to-soldier transition [17-19,34,39,40].
Here, we investigated the effects of specific hormone / semiochemical (JH III, JH
III+SHE, SHE) and socio-environmental conditions (live soldiers) on soldier caste
differentiation and gene expression by totipotent termite workers. Although there are
certainly other semiochemical and socio-environmental conditions that could play a
role in worker-to-soldier differentiation, we focused on the components listed above
because they build concisely on preceding work.
Phenotypic assay results were similar to past findings in that JH III induced
presoldier formation, JH III+SHE synergistically increased presoldier formation, and
SHE alone had no effect on presoldier development . The addition of live soldiers
to the bioassay did not impact soldier formation. Because presoldier differentiation
only occurs naturally in larger groups of workers over longer periods of time (see Fig.
1B and ), our small-scale model bioassay cannot allow for determination of any
inhibitory effects by soldiers at the whole organism level. Nonetheless, our results
provide good support to the hypothesis that SHE, or a component of it, acts with JH as
a primer pheromone to help regulate caste proportions within termite colonies.
This research also monitored phenotypic effects in concert with the expression
patterns of multiple genes. This was accomplished with destructive sampling of some
assay replicates for RNA isolation, while allowing others to proceed without
disturbance. The typical worker-to-presoldier differentiation process takes
approximately 15 days. To capture potential expression changes up to apolysis, gene
expression levels were monitored at 1, 5, and 10 days post treatment, which are
considered early, middle and late time points, respectively, in the presoldier
developmental transition. A total of forty-nine genes were investigated across three
replicate colonies. Statistically significant genes that passed the FDR cutoff were
clustered together based on expression pattern (Fig. 2,3,4). As discussed below, three
main groups of responsive genes were identified: (i) chemical production / degradation,
(i) hemolymph protein coding, and (iii) developmental.
Chemical production / degradation genes
Chemical production and degradation genes code for enzymes that are potentially
responsible for the production and / or degradation of many types of semiochemicals in
termites, including hormones such as JH and ecdysone, as well as the soldier head
terpenes y-cadinene and y-cadinenal. The three groups of genes included in this
category are cytochrome P450, hydrolytic, and mevalonate pathway protein-coding
Cytochrome P450s are known for their role in the oxidation of endogenous and
xenobiotic substrates including hormones, pheromones, insecticides, and secondary
plant compounds [41,42]. Specifically, P450s have been shown to play a role in the
biosynthesis and metabolism of morphogenic hormones (JH, ecdysone) and terpenoids
. On Day 1, two groups of P450s were differentially expressed. In the first group
(IIA), CYP15FI, CYP4C48, CYP6G? and CYP4C47 were down-regulated with JH III
and JH III+SHE treatments, while in the second group (I), CYP4C46 and CYP4U3 were
up-regulated with JH III and JH III+SHE treatments. This opposite expression profile
of the two P450 groups suggests they have different functions, likely acting on multiple
Past research has identified P450s that play significant roles in JH biosynthesis
and degradation in insects. In the cockroach, Diploptera punctata, CYP15A] epoxidizes
methyl farnesoate to form JH III . In the present study, those P450s that were
down-regulated with JH treatment (CYP15FI, CYP4C48, CYP6G? and CYP4C47)
could have a similar function. Insect P450s have also been shown to play a role in the
degradation of JH III, as is the case with CYP4C7, which converts JH III to 12-trans-
hydroxy JH III in Diploptera punctata [44,45]. The group I P450s (CYP4C46 and
CYP4U3) that were up-regulated in the present study could be playing this role and / or
the group of genes that were down-regulated could be inactivated, potentially blocking
the worker-to-soldier transition.
Juvenile hormone metabolism is also potentially mediated by hydrolytic
enzymes, including JH esterases and epoxide hydrolases . Three genes having
homology to JH esterases and epoxide hydrolases displayed significant expression
differences among treatments. Carbx-1 has highest homology to a JH esterase of the
wood-feeding beetle P'a, ,'ih a hilaris (BAE94685) . The Carbx-2 gene has
highest homology to a JH esterase of the sawfly Athalia rosae (BAD91555). Both the
Carbx-1 and Carbx-2 genes also have significant homology to honey bee JH esterases
 as described by Mackert et al. . Both genes are expressed in the gut, and thus
could be acting on JH acquired via trophallaxis, but also could play digestive roles by
hydrolyzing lignin or hemicellulose carboxyl esters .
Epoxide hydrolases are known to degrade JH by hydrolyzing the epoxide bond
that is formed by CYP15 action as described above. The epoxide hydrolase studied
here, Epox-1, has significant homology to an Aedes Cat ,lvli epoxide hydrolase
(XP_001651935), among others. If Epox-1 is acting as a JH epoxide hydrolase, its
observed up regulation could contribute to the degradation or inactivation of any
endogenous remaining JH prior to apolysis or ecdysis, which is expected to occur at
around day 10 in our model presoldier induction assays .
The production of JH and other sesquiterpenes derived from the mevalonate
pathway is important to termite colony success, not only for development and caste
differentiation, but also for production of defensive chemicals and pheromones that
possess a sesquiterpene backbone [29,50]. Both up- and down-regulation of genes in
the mevalonate pathway can significantly impact the production of JH and pheromones
[30,51]. In the present study, five mevalonate pathway genes were investigated: Famet-
1, Famet-2, Famet-3, Mev-1, and HMG. Two genes homologous to famesoic acid
methyl transferases (Famet-1, Famet-2) showed differential expression. Farnesoic acid
methyl transferase methylates farnesoic acid, producing the immediate JH precursor
methyl farnesoate . RNAi-mediated knockdown of this gene in Tribolium
castaneum has led to reduced JH levels and precocious molting . R. flavipes Famet-
1 shares strongest homology to a FAMet protein from the hymenopteran Melipona
scutellaris (AM493719) . Our results revealed that JH causes increased Famet-1
expression. Increased expression of this gene could theoretically increase JH
biosynthesis rates and enable soldier formation. Our results also revealed that the
presence of live soldiers down-regulates Famet-2 gene expression, which theoretically
could lead to reduced JH production and decreased worker-to-soldier differentiation.
In general, these results suggest that JH III causes up-regulation of mevalonate
pathway genes, while live soldiers are suppressive. Consistent with our phenotypic
bioassay results, suppression of the mevalonate pathway by live soldiers would likely
result in reduced pathway products, such as JH, resulting in reduced JH titers and
subsequent reductions in soldier caste differentiation.
Hemolymph protein coding genes
Four hemolymph protein coding genes, Hex-1, Hex-2, Vit-1, and Vit-2 showed
significant differential expression through all assay days. These four genes are
important in caste differentiation and sociobiology for a number of social insects;
therefore, it was not surprising that they showed responsiveness in our experiments.
The termite hexamerin genes have been shown to act as part of an environmentally
responsive socio-regulatory mechanism that affects the activity of JH, possibly limiting
its availability [18,33,34,54].
Two other hemolymph protein genes, Vit-1 and Vit-2, were up-regulated with
JH and JH +SHE treatments at Day 5, but only Vit-2 was differentially expressed at
Day 10. Throughout the experiment, both Vit-1 and Vit-2 genes displayed a high
amount of variability among treatments and replicates. One explanation for such
variance is the inclusion of both sexes of worker termites in assays. In most insects,
vitellogenin (Vg) serves as a female-specific yolk precursor protein that functions in
oocyte provisioning. However, Vg has also been shown to play a role in social insect
caste regulation; for example, Vg in female honeybee workers, has been shown to
interact with JH. Specifically, higher JH levels and lower Vg levels increased the
transition from nursing to foraging behavior by worker bees , while a reduction of
JH delayed the onset of foraging . Honeybee workers with RNAi-suppressed Vg
levels performed foraging behaviors earlier than untreated workers [36,37]. Nutrition
has also been shown to affect Vg and JH by regulating the transition from nursing to
foraging . Finally, Vg has been shown to affect queen honeybee longevity by
interacting with insulin signaling . Together, these findings suggest that honeybee
Vg has been co-opted away from reproduction to serve as a regulator of caste
behavioral polyethism . Results of the current study, showing that Vit-1 and Vit-2
are up-regulated with JH and JH +SHE treatments, but down-regulated with SHE and
live soldier treatments, suggest interesting possibilities with respect termite vitellogenin
and caste polyphenism.
The dramatic morphological change that occurs as worker termites become
soldiers requires significant body plan rearrangement . The soldier termite's large
mandibles and their associated muscles represent a large change from the smaller head
and reduced muscle mass present in worker termites [58,59]. Thus, it is likely that
multiple genes are required to coordinate and achieve this transition . Six
developmental genes from two groups, cytoskeletal/ structural and body-plan, showed
significant differential expression in the current study.
The cytoskeletal/ structural protein coding gene "fl-tube" was significantly
differentially expressed at Day 1 among treatments. f-tubulins are also hormone-
responsive and have been linked to the production of ecdysteroids in Manduca sexta
[61,62]. a- and f-tubulin genes were also identified in Bombyx mori from several EST
libraries linked to imaginal wing disk metamorphosis and 20-hydroxyecdysone [63,64],
suggesting roles in restructuring during adult wing formation. Our findings suggest
potential roles for R. flavipes fl-tube in either soldier head muscle function or possibly
ecdysone-linked developmental-regulatory processes.
A number of developmental/ body plan genes also showed significant differential
expression. One body plan gene, broad (BTB/POZ) , which is homologous to broad
(br) transcription factor genes of the hemimetabolous and holometabolous insects
(Oncopeltusfasciatus and T. castaneum), was up-regulated at Day 5 with JH and
JH+SHE treatment and at Day 10 with JH+SHE treatment. Erezylimaz et al.  used
RNAi to silence the br gene in 0. fasciatus, causing an additional immature molt.
Erezylimaz et al. suggested that br is required for morphogenesis, and that its
expression is regulated by JH. RNAi silencing of br in T. castaneum caused similar
results . If br is acting in the same manner in termites, up-regulation of the gene by
JH+SHE would promote the worker-to-soldier transition, which is in agreement with
phenotypic bioassay results showing increased presoldier formation in the JH+SHE
The research presented here demonstrates for the first time the influence that the
SHE blend, live soldier caste members, and JH together have on phenotype and gene
expression of totipotent termite workers (Fig. 6). To summarize phenotypic assay
results (Fig. 6A): (i) JH III induced significant presoldier differentiation, (ii) JH + SHE
induced significantly higher levels of presoldier differentiation, (iii) the crude SHE
blend by itself did not have any observable phenotypic effects, and (iv) live soldiers
inhibited presoldier formation in the absence of ectopic JH. In support of primer
pheromone hypotheses initially proposed by Liischer  and further developed by
Henderson , our results provide the first evidence that the soldier caste has direct
impacts on caste-regulatory gene networks, and subsequently, worker caste
differentiation. Significant responsive gene categories identified here include chemical
production / degradation genes, hemolymph protein coding genes, and developmental
genes (Fig. 6b). Past reports (e.g., ) and the present research (Fig. 1B) have
demonstrated that live soldiers do indeed inhibit natural presoldier formation. These
results, in addition to the current gene expression findings, support earlier hypotheses
that live soldiers act as part of a negative feedback loop, inhibiting new soldier
formation by regulating the expression of genes important for caste differentiation (Fig.
6b) [16,24,68]. Recent findings have further revealed that y-cadinene and y-cadinenal
levels increase in workers that are held with soldiers (MR Tarver, unpublished results),
which lends significant strength to the results presented here that show live soldier and
SHE impacts on gene expression. The next steps in this research will follow up on
these observations by investigating the impacts of pure y-cadinene and y-cadinenal on
phenotypic caste differentiation and on the expression of responsive genes identified in
the current study.
This research provides important new evidence of impacts on nestmate gene
expression by live termite soldiers and crude soldier head extracts. While further
research is needed to resolve the roles of soldiers and SHE blend components in termite
caste regulation (via RNA interference, gene expression localization, further analysis of
SHE constituents, investigating impacts of SHE constituents on gene expression, or
using whole-genome micro-arrays or next-generation transcriptome sequencing) the
findings of this study provide a solid foundation on which to conduct further
R. flavipes colonies were collected from different locations near Gainesville,
Florida USA. Termites were held in the laboratory for at least two months before use in
bioassays. Colonies were maintained in darkness within sealed plastic boxes, at 220C.
All colonies contained male and female neotenic reproductive. Termites were
considered true workers if they did not possess any sign of wing buds or distended
abdomens. Termites were identified as R. flavipes by a combination of soldier
morphology , and 16S mitochondrial-ribosomal RNA gene sequencing . The
partial mitochondrial 16S sequences of the four colonies used were deposited,
respectively, in Genbank under accession numbers: FJ265704 (colony-1 "GB 1"),
FJ627943 (colony-2 "K2"), FJ265705 (colony-3 "A8") and GQ403073 (colony-4
"K5"). Using the 16S mitochondrial sequences, colony 1 was 99% identical to
mitochondrial haplotypes F22 and Fl (EU259755, EU259734), colony 2 was 98%
identical to haplotype F20 (EU259753), colony 3 was 96% identical to haplotypes F34,
28, and 21 (EU259767, EU259761, EU259754) and colony 4 was 98% identical to
haplotype F20 (EU259753).
Small-scale dish bioassays were conducted at 270C as described previously
[19,39]. Paired paper towel sandwiches were treated with acetone (controls), JH III, or
SHE treatments delivered in acetone. JH III (75% purity; Sigma; St. Louis, MO) was
provided at a rate of 112.5 pg per dish in a volume of 200 p1 acetone. The JH III rate
was chosen based on maximal efficacy with minimal mortality observed in previous
concentration range studies . After solvent evaporation, paper towel sandwiches
were placed in 5 cm plastic Petri dishes and moistened with 150 1p of reverse osmosis
water. Fifteen worker termites were placed in each assay dish. Live solider treatments
consisted of holding two live soldiers with 15 workers from the same colony. Every
five days, termites were counted, presoldier formation was noted, and water was added
if needed. Each treatment was monitored for 25 days.
For larger-scale soldier inhibition assays, two treatments were examined to assess
the influence of live solders on presoldier formation in large group format of 100
termites per dish. Control treatments included 100 workers only; whereas, soldier
treatments included 90 workers + 10 soldiers. Termites from a single colony (K5) were
used, and assays were run in large Petri dishes (9 cm diam.). Caste composition and
survival were monitored every ten days for a total of 30 days. Each treatment was
replicated five times and results pooled (avg SEM) for reporting.
Preparation of solider head extracts
Soldier head extracts were prepared as described in Tarver et al. . Soldiers
(80-150 total) were isolated from lab colonies, and their heads removed and
homogenized in 5 mL acetone using a Tenbroeck glass homogenizer. To remove
particulate matter, the homogenate was fractionated by passing it through a glass
Pasteur pipette filled with approximately 250 mg of silica gel (60-200 mesh) on top of a
glass wool plug. The SHE was eluted with 10 column volumes of acetone and brought
to 50 ml with acetone in a volumetric flask.
Gene expression bioassays
A total of five different treatments were tested including acetone controls (300
a1), JH III (200 11 acetone containing 112.5 pg JH III), JH III+SHE (112.5 pg JH III in
acetone + 1.5 soldier head equivalents in acetone), SHE (1.5 head equivalents extracted
in acetone), and live soldiers (two per assay replicate). Each treatment was replicated
five times for colony-1 and six times for colonies-2 and 3 (GB1, K2, and A8
respectively). Three biological replicates were used per treatment for colony 1 and four
for colonies 2 and 3. Additional replicates for colonies 2 and 3 were added to improve
statistical power. Samples of 15 termites were collected for destructive sampling at
days 0, 1, 5, and 10. Collected samples were immediately frozen at -800C.
RNA isolation and cDNA synthesis
Total RNA was isolated from frozen samples using the SV total RNA Isolation
System (Promega; Madison, WI) according to the manufacturer's protocol. Whole
body RNA extracts were isolated from all 15 worker termites included in each bioassay
dish. The amount of RNA was quantified by spectrophotometry and equal amounts of
RNA were used in cDNA synthesis reactions. First-strand cDNA was synthesized using
the iScript cDNA synthesis Kit (Bio-Rad; Hercules, CA) according to the
The 49 candidate and reference genes were identified in recent R. flavipes
sequencing projects and were chosen based on their homology to developmental or JH
biosynthesis / metabolism genes [8,17,34,40,54,71]. The identity of all 49 PCR
products was verified by direct sequencing. Quantitative real-time PCR (qRT-PCR)
was performed using the iCycler iQ real-time PCR detection system (Bio-Rad) with
SYBR-green product tagging (similar to [8,34]). cDNA, obtained as described above,
was used as the qRT-PCR template. Gene specific primers are listed in Additional file
2: Table S2. Eleven total biological replicates were conducted for qRT-PCR (three
from colony-1, and four each from colonies 2 and 3). Average Ct values of three
technical replicates were pooled for analysis to represent each biological replicate.
Reference gene selection
To select appropriate reference genes, all of the Ct values across all colonies,
treatments, biological replicates, and technical replicates for each gene were analyzed
to identify genes with the least amount of variation in expression (see [17,34]). Three
genes with the lowest standard deviation were chosen for use as reference genes: Stero-
1, LIM, and Mev-1 (Additional file 1: Table Si).
Data and statistical analyses
Relative expression of target genes was calculated by comparing the average of
the three technical replications first normalized to the reference genes and then
normalized to the control treatment using the 2-ActAct method . Normalized
expression values (2-ActAct) from all colony replicates were initially analyzed using the
microarray visualization software ArrayStar (DNASTAR, Inc, Madison, Wisconsin,
USA). To identify potential gene networks, genes with significant differential
expression were clustered hierarchically using Euclidean distance metrics and centroid
linkage for each day (1, 5 and 10) using ArrayStar.
To identify similarities between treatments, all genes were clustered
hierarchically using Euclidean distance metrics and centroid linkage for each day (1, 5
and 10) using Array StarTM software.
To determine significantly differentially expressed genes, CT expression values
for target genes were normalized to the CT values from the reference genes (ACT). A
two-way ANOVA, with adjusted least squares (LS) means and false discovery rate
(FDR) correction was used to distinguish significantly differentially expressed genes
among treatments using JMP statistical software (SAS Institute, Cary, NC, USA)
(Additional file 3: Table S3). Tukey's HSD test was used for separating means by
treatment for each gene.
MRT conceived the study design, performed all the experimental procedures and was
the primary author of the manuscript. XZ participated in the design of the study. MES
conceived the study design, analyzed data, and critically revised the manuscript. All the
authors read and approved the final manuscript.
We thank Aur6lien Tartar for assistance with gut gene bioinformatics, Daniel Hahn for
valuable discussions on expression analysis, and Alan Lax and Dunhua Zhang for
critical reading of manuscript drafts. This work was supported by CSREES-USDA-NRI
grant No. 2007-35607-17777 to MES and XZ, by The Consortium for Plant
Biotechnology Research, Inc. and DOE Prime Agreement No. DE-FG36-02GO12026
to MES, and a University of Florida IFAS Innovation Grant to MES.
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Fig. 1. Impact of semiochemical and socio-environmental treatments on
soldier caste differentiation. (A) Cumulative presoldier formation through Day 25
of assays that compared five different treatments: untreated controls, JH III, JH
III+SHE, SHE, and live soldiers for three different colonies. Each replicate dish (n=5
per colony) contained 15 workers. Results for the three colonies were pooled for
analysis. Adjusted LS means are shown; bars with the same letter are not significantly
different (P<0.05). (B) Post-hoc presoldier induction assays conducted using 100
termites per replicate dish (n=5). The two treatments that were examined included
starting compositions of 100 workers + 0 soldiers, or 90 workers + 10 soldiers. No
presoldiers formed after 30 d. in treatments that included soldiers at the beginning of
assays. This finding reveals that R. flavipes soldiers indeed are capable of inhibiting
worker differentiation to presoldiers.
Fig. 2. Expression changes for significant genes in termite workers in
response to hormonal, semiochemical and socio-environmental
treatments after 1 day. Results shown represent the relative expression values of
significant differentially expressed genes under five different treatments: control, JH
III, JH III+SHE, SHE, and live soldiers after one day; Blue boxes represent genes that
are down-regulated while red boxes represent genes that are up-regulated. Boxes with
the same letter within a row are not significantly different (FDR). Dendrograms at the
left group genes by similar expression pattern.
Fig. 3. Expression changes for significant genes in termite workers in
response to hormonal, semiochemical and socio-environmental
treatments after 5 days. See Fig. 2 caption for details.
Fig. 4. Expression changes for significant genes in termite workers in
response to hormonal, semiochemical and socio-environmental
treatments after 10 days. See Fig. 2 caption for details.
Fig. 5. Hierarchical clustering results of gene expression pattern
clustered by treatment. All three days (A- Day 1, B- Day 5, and C- Day 10) were
analyzed separately using the relative expression of each gene by the Euclidean
distance metric, with centroid squared linkage.
Fig. 6. Diagrams summarizing the influence of socio-environmental and
semiochemical factors on caste differentiation. A) Semiochemical and socio-
environmental factors tested and their effects on worker-to-soldier differentiation. JH
III and JH+SHE caused an increase in soldier formation, while SHE had no effect on
presoldier/soldier formation. Past research [15,16, personal observations] indicates that
soldiers inhibit worker differentiation. B) Diagram representing how socio-
environmental and semiochemicals factors might modulate the expression patterns of
multiple genes and caste differentiation. Networks including the following gene
categories showed significant changes among treatments: chemical
production/degradation, hemolymph protein coding, and developmental genes. Dotted
lines represent the possible feedback loop when colony worker termites molt into
soldiers, the increase in the soldier number consequently inhibits the formation of
Additional file 1
Title: Table S1
Description: Meta-analysis of all genes used to identify reference genes
having the most stable expression. Genes highlighted in yellow are those
with the most stable expression across time and treatments (i.e., smallest
standard deviation; SD).
Additional file 2
Title: Table S2
Description: Sequence accession numbers and quantitative real-time
PCR primer details
Additional file 3
Title: Table S3
Description: Summary of ANOVAs for each gene (down) and day (across).
Additional file 4
Title: Table S4
Description: Day 1 relative expression values and summarized ANOVA
results with FDR q-values
Additional file 5
Title: Table S5
Description: Day 5 relative expression values and summarized ANOVA
results with FDR q-values
Additional file 6
Title: Table S6
Description: Day 10 relative expression values and summarized ANOVA
results with FDR q-values
Additional file 7
Title: Table S7
Description: Summary of horizontal gene clustering for Figures 2, 3 and
- Worker %
1 Soldier %
day 0 day 30
day 30 Assay Days
Control JH-llI JH+SHE SHE Live Soldiers
b b b b Carbx-1
b b ab b Myosin
b b b ab Bactin
ab b a ab ab Btube
ab b ab ab a R-Pro
2 ab b ab ab a ATPase
ab b b ab HMG
ab b ab a Hex-2
ab b b Vab 18s
a a a a NADH
II ab a a ab nanos
b I b b CYP15F1
b b b CYP4C48
Sbc c bc CYP6G?
b b b b CYP4C47
a a a CYP4C46
Figure 2 a a ab CYP4U3
SHE Live Soldiers
Day 10 Control
Live Soldiers SHE JHIII
Live Soldiers SHE JHIII
I Social environment I I
JH+SHE -- Soldiers
molymph protein codi
Additional files provided with this submission:
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Additional file 2: Additional File 2.doc, 277K
Additional file 3: Additional File 3.doc, 1325K
Additional file 4: Additional File 4.doc, 313K
Additional file 5: Additional File 5.doc, 313K
Additional file 6: Additional File 6.doc, 306K
Additional file 7: Additional File 7.doc, 238K